Cytotoxic 13,28 Epoxy Bridged Oleanane-Type Triterpenoid Saponins from the Roots of Ardisia crispa

Ardisiacrispin D–F (1–3), three new 13,28 epoxy bridged oleanane-type triterpenoid saponins, together with four known analogues (4–7) were isolated from the roots of Ardisia crispa. The structures of 1–7 were elucidated based on 1D and 2D-NMR experiments and by comparing their spectroscopic data with values from the published literatures. Ardisiacrispin D–F (1–3) are first examples that the monosaccharide directly linked to aglycone C-3 of triterpenoid saponins in genus Ardisia are non-arabinopyranose. In the present paper, all compounds are evaluated for the cytotoxicity against three cancer cell lines (HeLa, HepG2 and U87 MG) in vitro. The results show that compounds 1, 4 and 6 exhibited significant cytotoxicity against Hela and U87 MG cells with IC50 values in the range of 2.2 ± 0.6 to 9.5 ± 1.8 µM. The present investigation suggests that roots of A. crispa could be a potential source of natural anti-tumor agents and their triterpenoid saponins might be responsible for cytotoxicity.


Introduction
The genus Ardisia (Primulaceae) is widely distributed in subtropical and tropical regions of the world, and, for along time, its roots have primarily been used as traditional medicines [1]. In previous phytochemical investigations, the chemical constituents of the genus Ardisia were reported to be saponins, isocoumarins, peptides, quinones and alkylphenols [1]. The roots of Ardisia species appeared to be a rich source of triterpenoid saponins, which have been isolated from various Ardisia species, including A. crenata, A. crispa, A. mamillata and A. pusilla. For example, novel triterpenoid saponins, ardisicrenosides A-B and ardisiacrispins A-B, were previously isolated from A. crenata and A. crispa, respectively [1]. Phytochemical investigations revealed that triterpenoid saponins are the main constituents of the genus Ardisia, and have significant cytotoxic activities [2][3][4][5][6][7][8]. Triterpenoid saponins reported from Ardisia plants are interesting from the viewpoints of chemical diversity and biological activity.
The roots of Ardisia crispa are utilized as traditional Chinese medicine for treating a sore throat, damp-heat jaundice and bruises [9]. Previous pharmacological studies on this plant have revealed that it has anti-tumor, anti-inflammatory and suppressed angiogenesis effects [10][11][12][13][14][15][16][17][18], but its chemical composition is rarely reported. In order to find more potentially cytotoxic triterpenoid saponins from A. crispa, an extract of the roots of this species was chemically investigated. Herein, the isolation, structural elucidation and the cytotoxic activities of the triterpenoid saponins are discussed.

Results and Discussion
Compound 1 was obtained as a white amorphous powder. Its molecular formula was deduced as C 41 3 -29); and 2 oxygen-bearing methylene protons δ H 3.55 (1H, d, J = 11.1 Hz, H-30a), 3.25 (1H, d, J = 11.1 Hz, H-30b), together with 2 anomeric proton signals δ H 5.23 (1H, brs, H-1 ) and δ H 4.44 (1H, d, J = 7.8 Hz, H-1"). The above 1 H-NMR data, together with a carbonyl carbon signal at (δ C 181.0, C-28) and an oxygenated quaternary carbon at (δ C 95.0, C-13) in the 13 C-NMR spectrum, suggested 1 to be a 13,28 epoxy bridged oleanane-type triterpenoid skeleton in the aglycone and the presence of 2 sugar units [19]. The 13 C-NMR data of the aglycon in 1 was similar to ardisicrenoside A, which was obtained previously from the Ardisia crenata, except for the presence of a carbon signal of γ-lactone moiety at C-28 (δ C 181.0) in 1, rather than a oxygenated methylene carbon signal (δ C 77.5) in ardisicrenoside A [19]. The identity of the monosaccharides and the linkage of the sugar residues were made by the combination of DEPT-135, HSQC, 1 H-1 H COSY and HMBC spectra. The connectivity of the 2 sugars was mainly based on the HMBC correlations: H-1 (δ 5.23, 1H, brs) with C-3 (δ C 89.4) of the aglycone, H-1" (δ H 4.44, 1H, d, J = 7.8 Hz) with C-2 (δ C 89.6) ( Figure 1). By comparing the chemical shift signals and coupling constants of the sugar moieties from the published literature [20], the relative configurations of the anomeric centers of the arabinosyl and glucosyl moieties in 1 were determined to be α and β, respectively. The two kinds of sugars, L-arabinofuranose and D-glucose, were identified by GC analysis after derivatization.   Compound 2 was separated as a white amorphous powder. The molecular formula of 2 was established to be C 42 H 70 O 14 from its HR-ESI-MS at m/z 821.4628 [M + Na] + (calcd. for 821.4657). In the 1 H and 13 C-NMR spectrum of 2 (Table 1), signals due to an aglycone moiety were similar with those of ardisicrenoside B [19], although the signals due to the sugar moiety were not identical. The NMR data of 2 showed two anomeric proton signals at δ H 4.39 (1H, d, J = 7.8 Hz, H-1 ) and 4.65 (1H, d, J = 7.7 Hz, H-1"), corresponding to two anomeric carbons at δ C 105.9 and 104.6 in the HSQC spectrum, indicating the presence of two sugar moieties. The attachments of the sugar chain were deduced from the HMBC spectrum. The HMBC showed that there was a correlation between H-1 (δ H 4.39, 1H, d, J = 7.8 Hz) and C-3 (δ C 91.3) of the aglycone; H-1" (δ H 4.65, 1H, d, J = 7.7 Hz) and C-2 (δ C 79.1) indicated that the sugar chain was connected to C-3 of the aglycone, and the terminal sugar was linked to C-2 . The anomeric configuration of these 2 glucoses were determined to be β on the basis of the J value of the anomeric proton in glucose (J = 7.8 and 7.7 Hz)]. The relative configurations of 2 were determined by the NOESY experiment, which were consistent with those of 1. On the basis of above evidence, the structure of 2 was identified to be 3β, 16α, 30-trihydroxyolean-13β,28-epoxy-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranose, and named as ardisiacrispin E. Table 1. 1 H and 13 C-NMR data of compounds 1-3 in methanol-d 4 (δ in ppm).

2 3
Position  (Table 1), except for 13 C-NMR signals of C-20 (δ C 36.9), C-29 (δ C 28.5) and C-30 (δ C 66.5) in 2, which were shifted to δ C 49.2, 24.3 and 209.2 in 3, respectively. Moreover, the singlet observed at δ H 9.40 was assigned as a formyl proton. The above NMR data suggested that C-30 in 3 was substituted with a formyl group, which was supported by the HMBC correlations from H-29/H-30 to C-20 and from H-29 to C-19, C-21 and C-30. The NMR data for the sugar moiety and GC analysis of the derivatives of the hydrolyzate of 3 indicated the existence of D-xylopyranose and D-glucopyranose. The anomeric proton signals of 3 at δ with δ C 80.9 (C-2 ) were observed. The 1 H and 13 C-NMR spectra of the sugar parts of 3 were in accordance with those of majonoside-R2 [21]. The relative configurations of 3 were determined by the NOESY experiment, which were consistent with those of 2. Therefore, the structure of 3 was identified as 3β, 16α dihydroxyolean-13β,28-epoxy-30-al-3-O-β-Dxylopyranosyl-(1→2)-β-D-glucopyranose, and named as ardisiacrispin F.
To date, the monosaccharide directly linked to aglycone C-3 of triterpenoid saponins reported in Ardisia are all arabinopyranose. In this study, we reported three novel triter-penoid saponins from Ardisia crispa with arabinofuranosyl and glucosyl groups directly connected to aglycone C-3 for the first time. Therefore, the above results are of great significance to elucidate the biosynthesis of triterpenoid saponins in genus Ardisia.
In addition, all the isolates (1-7) were tested for their cytotoxicity against the HeLa, HepG2 and U87 MG cell lines using the MTT method. The results ( Table 2) show that the compounds (1, 4 and 6) exhibited more significant cytotoxic activities than cisplatin (Sigma, >99%), which was used as a positive control. Due to the limited numbers of active compounds, only the superficial and primary structure-activity relationships were discussed. Compared with compounds 4-7, 1-3 showed stronger cytotoxicity against Hela cells, which suggested that the non-arabinopyranose directly linked to aglycone C-3 might make a contribution to their cytotoxicity. However, The IC 50 values of compounds 2 and 3 on HepG2 cells were higher than 4-6, which might be caused by the arabinopyranosyl group in 4-6. Compound 5 exhibited better cytotoxic activity against U87 MG cells compared with 1-4 and 6-7, which demonstrated that the number of monosaccharide may affect the activity. Table 2. Cytotoxic activities of compounds 1-7 against three human tumor cell lines (HeLa, HepG2 and U87 MG) (mean ± SD, n = 3).

General Experimental Procedures
Optical rotations were measured on a JASCO P-2000 instrument. The 1D and 2D NMR spectra were recorded on either a Bruker DPX 400 instrument with tetramethylsilane as an internal standard and MeOH-d 4 as a solvent. HR-ESI-MS experiments were conducted using a Thermo Fisher QE Focus spectrometer. The semi-preparative HPLC procedure was conducted on a Shimadzu LC-16D instrument with an RID-20A (reflective index detector) and a reversed-phase C 18 column (250 × 10 mm, 5 µm, Waters SunFire). Column chromatography was performed using silica gel (200-300 mesh, China), octadecyl silica (ODS) (50 µm, Merck, Germany).

Plant Material
The roots of Ardisia crispa were collected from Kaili of Guizhou Province (China), and identified by Professor Sheng-Hua Wei from Guizhou University of Traditional Chinese Medicine. The voucher specimen (No. 20190502) was deposited at Guizhou University of Traditional Chinese Medicine.

Acid Hydrolysis of Ardisiacrispin D-F (1-3)
In order to determine the absolute configuration of the monosaccharide in triterpenoid saponins, the acid hydrolysis of new compounds were performed. The acid hydrolysis of ardisiacrispin D-F (1-3) (1.0 mg each) was performed according to the previous literatures [20,25,26]. The trimethylsilylthiazolidine derivatives from the n-hexane layer were analyzed with GC-MS with a DB-5 capillary column. The absolute configurations of the sugar moieties were established by comparison with the retention times of the authentic sugars (D-xylose, 15.56 min; L-arabinofuranose, 16.40 min; D-glucose, 18.21 min).

Cytotoxic Activity
The cytotoxic activity of compounds (1-7) against three human cancer cell lines, namely Hela (human cervical cancer cells), HepG-2 (human hepatoma cells) and U87 MG (human glioblastoma cells), were evaluated by the MTT colorimetric assay described in a previous paper [27]. All the cells were cultured in DMEM supplemented with 10% FBS and antibiotics in a humidified atmosphere containing 5% CO 2 at 37 • C. Briefly, 100 µL of adherent cells were seeded into each well of 96-well cell culture plates at optimal cell density (1 × 10 5 cells per well) and allowed to adhere for 12 h. The IC 50 value of each compound was tested on the basis of cell viability after 48 h of treatment with different concentrations of compounds, with cisplatin (Sigma, >99%) as the positive control.

Conclusions
To summarize, a total of seven 13,28 epoxy bridged oleanane-type triterpenoid saponins were isolated from the roots of A. crispa and 3 of them were new structures. In previous investigations, Ardisia species appeared to be a rich source of triterpenoid saponins and have significant cytotoxic activities. In this paper, compounds (1-7) were evaluated for the cytotoxicity against three cancer cell lines (HeLa, HepG2 and U87 MG) in vitro. Compounds 1, 4 and 6 exhibited significant cytotoxicity against Hela and U87 MG cells with IC 50 values in the range of 2.2 ± 0.6 to 9.5 ± 1.8 µM. The present investigation suggested that the roots of A. crispa could be a potential source of natural anti-tumor agents. Their triterpenoid saponins might be responsible for cytotoxicity, and also seem to be of great chemotaxonomic value for A. crispa.